US6824985B1 - Formulation for reducing urea effect for immunochromatography assays using urine samples - Google Patents
Formulation for reducing urea effect for immunochromatography assays using urine samples Download PDFInfo
- Publication number
- US6824985B1 US6824985B1 US08/925,998 US92599897A US6824985B1 US 6824985 B1 US6824985 B1 US 6824985B1 US 92599897 A US92599897 A US 92599897A US 6824985 B1 US6824985 B1 US 6824985B1
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- United States
- Prior art keywords
- urea
- test sample
- analyte
- strip
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related, expires
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/97—Test strip or test slide
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/81—Tube, bottle, or dipstick
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/825—Pretreatment for removal of interfering factors from sample
Definitions
- a common format for these tests involves the use of an immunostrip.
- this format involves a matrix of a material through which a fluid test sample, such as urine, can flow by capillarity.
- the matrix typically in the form of a strip through which the test fluid flows horizontally, contains an antibody specific for the analyte whose presence and/or concentration is being sought which antibody bears a detectable label, so that the presence and/or concentration of the analyte in the test fluid can be determined by detection of the signal emitted from the detectable label.
- FIG. 1 A classical embodiment of such a device, sometimes referred to as an immunochromatographic strip, is illustrated by FIG. 1 .
- strip 10 bears a labeled antibody, specific for the analyte under investigation, in zone 13 which binds with the analyte in the fluid test sample applied to the wicking zone 12 of the strip 10 which may contain a buffer and flows along the strip to form an immunocomplex which further migrates due to capillary action through the capture zone of the strip 14 and the optional detection zone 16 .
- the capture zone 14 there is immobilized the analyte or a derivative thereof which is immunoreactive with the antibody and is able to capture labeled antibody which has not reacted with analyte in the fluid test sample.
- Detection zone 16 is optional but can contain immobilized anti-mouse IgG to bind the analyte/labeled binding partner complex and thereby serve as a means for verifying that the test has been carried out correctly.
- Urine is a common test fluid for this sort of assay, and it has now been discovered that urea, whose concentration in urine will vary tends to interfere with the binding between the antibody and analyte in the system. The degree of such interference will vary depending on the particular antibody and antigen involved in the assay.
- Urea is synthesized in the liver as the final product of amino acid catabolism. Urea excretion is proportionate to protein intake and dependent on the protein metabolism, it increases with a high protein diet. Normal adults excrete about 20.6 g of urea per day.
- the interferences with the antigen/antibody binding tend to cause the assay to have a negative bias when the urine test sample contains more than the normal amount of urea. It would be desirable, and it is an object of the present invention to provide a method and a device which reduce or eliminate the bias caused by the fluctuations in urea concentration in urine.
- the present invention is a method for the analysis of an analyte in a urine test sample in which a labeled antibody specific to the analyte is combined with the urine and the concentration of analyte in the urine is determined by the magnitude of the response from the labeled antibody which is a function of the amount of binding of the labeled antibody with the analyte.
- the assay is improved upon by maintaining the concentration of urea in the urine above a threshold value which is the concentration at which increases in urea do not affect the binding between the analyte and the labeled antibody.
- a test device for carrying out the improved assay.
- FIG. 1 represents a type of test strip which is suitable for use in carrying out the present invention.
- FIG. 2 graphically illustrates the bias introduced to an analysis of urine by raising the urine's specific gravity.
- FIG. 3 graphically illustrates the plateau of the urea interference in urine assays when the urea concentration reaches 225 mM.
- FIG. 4 illustrates the discovery that the addition of urea to an assay of urine can limit the bias caused by the urea effect.
- the assay improvement of the present invention can be carried out in a wet system such as a microtitre plate assay, it is typically employed in conjunction with an immunostrip format.
- the invention is practiced by first providing a test strip in the form of a matrix through which the test sample together with analyte contained therein and the labeled antibodies can flow by capillarity.
- the matrix will be in the form of a strip through which the test fluid flows horizontally, although the matrix could be set up in layers through which the test fluid would flow vertically from top to bottom or vice-versa.
- the following discussion focuses on the strip format.
- the strip can be prepared from any material through which the urine test sample, analyte and labeled antibody can flow by capillarity.
- suitable matrix materials include nitrocellulose, polysulfones, polycarboxylic acids, and filter paper.
- a particularly suitable immunochromatographic strip format for use in relation with the present invention is that which is disclosed in U.S. Pat. No. 4,446,232 wherein there is described a device for the determination of the presence of antigens which comprises a strip of a matrix material having a first zone in which there are provided enzyme linked antibodies specific to the analyte to be determined and a second zone containing immobilized analyte.
- a device for the determination of the presence of antigens which comprises a strip of a matrix material having a first zone in which there are provided enzyme linked antibodies specific to the analyte to be determined and a second zone containing immobilized analyte.
- the labeled antibodies can flow through the second zone when reacted with analyte introduced along with the test fluid but will not so flow in the absence of analyte in the test fluid due to their being bound in this zone by interaction with the immobilized analyte.
- the analyte is typically an antigen although the format can be designed to detect the presence of antibodies as analyte.
- An alternative to this format is that in which the capture zone contains an immobilized binding partner which is specific for an epitope of the analyte different than that to which the labeled antibody is specific.
- This format provides a means for capturing the labeled binding partner using the so-called sandwich format.
- the conjugate in a discrete detection zone located downstream on the strip from the zone in which the labeled binding partner for the analyte is bound, there are provided two zones from which the physically detectable property of the detectable label can be measured to determine its intensity and hence the concentration of the detectable label in a particular region of the strip.
- the accuracy of the final result can be skewed by changes of the urea concentration in the urine test sample caused by normal fluctuation in the urine's urea concentration and it is the goal of the present invention to reduce or eliminate this bias. This is accomplished by maintaining the concentration of urea in the urine test sample above a threshold value which is the concentration of urea at which increases in its concentration do not further affect the binding between the analyte and the labeled antibody.
- DPD deoxypyridinoline
- the three levels of DPD were 25, 75 and 250 nM and the urea concentrations were 75, 225, 400, 600 and 700 nM.
- the recovered DPD concentrations of these solutions were obtained from the standard curve generated using calibrators containing 400 mM of urea.
- the results shown in FIG. 3 indicate that the urea interference reaches a plateau at urea concentrations of greater than 225 mM.
- the urea concentration at which analytes other than DPD exhibit the plateau effect may be lower or higher than 225 mM, but can be readily determined for each analyte by routine experimentation such as that which is described herein for DPD. Accordingly, based on this discovery, which is the essence of the present invention, it is possible to factor out of the assay results bias caused by urea interference with antigen-labeled antibody binding in immunologic urinalysis. This is accomplished by combining at least the threshold level of urea with the urine test sample before it is contacted with the labeled antibody. In a liquid state assay, the necessary amount of urea can be combined with the urine sample or to the microtitre plate in which the labeled antibodies are disposed.
- the urea can be added to the strip's wicking pad or to a separate buffer pad included as an integral part of the strip. This is accomplished by impregnating the strip's pad with an aqueous solution of urea followed by drying, so that the urea will be made available by rehydration upon contacting the strip with the urine sample. Any of these methods will provide the necessary urea concentration. It is, however, preferred to combine the urea with the buffer pad to provide a unitary device which will automatically introduce the desired urea concentration to the urine sample upon contacting it with the strip. It is preferred to incorporate the urea into the buffer pad because it is in this area that the urine test sample first contacts the test device and combining the buffer and urea is the most efficient method for introducing the additional urea to the urine sample.
- analytes are present in urine which are determinable by means of the present invention.
- these analytes are deoxypyridinoline (DPD), human albumin, drugs of abuse, cancer markers, and human choronic gonatropin (hCG).
- the detectable label for the analyte may be any moiety which is detectable by reproducible means.
- the label can be an enzyme, a radioisotope, a chemilluscent material or a visible particulate label such as gold sol or latex particles.
- the present invention is further illustrated by the following example.
- buffer-urea pads 4′′ (10.2 cm) ⁇ 17.7′′(45 cm) of Whatman F075-07 membrane were prepared by hand impregnation.
- One formulation contained 1 M glycine for buffer at pH 8.6 and 300 mM urea.
- the second contained 1 M glycine at pH 8.6 and 400 mM urea.
- the third was used as a control and contained only 1 M glycine at pH 8.6.
- the buffer-urea pads were dried in a convection drier for 30 minutes at 40° C. The pad was then slit into 0.5′′ ⁇ 17.7′′ strips.
- the capture reagent was DPD-PEG, a poly(ethylene glycol) derivatized DPD, in 0.05 mg/mL concentration.
- the detection reagent was goat anti-mouse IgG in 0.3 mg/mL concentration.
- the striping of capture and detection reagents was done with an IVEK linear striper on Millipore nitrocellulose, 1′′ (2.54 cm) ⁇ 17.7′′ (45 cm).
- the strips were developed by dipping them in aqueous solutions containing 0, 20, 40, 80, 160 and 320 mMolar concentrations of DPD and either 75 mM or 600 mM of urea.
- the dose response decode was determined using the green filter on a CLINITEK 50® reflectance spectrometer.
- FIG. 4 The results of this experiment are graphically represented by FIG. 4 .
- those strips with 300 mM or 600 mM urea incorporated into the buffer pad gave dose response results which were grouped together for test solutions containing either 75 or 600 mM urea.
- the results obtained using the strip with no urea in the buffer pad are significantly different from the others.
- the responses of the immunoactivity are higher than those with total urea concentrations of 375, 900, 475 and 1000 mM.
Abstract
Description
Claims (10)
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/925,998 US6824985B1 (en) | 1997-09-09 | 1997-09-09 | Formulation for reducing urea effect for immunochromatography assays using urine samples |
CA002237269A CA2237269A1 (en) | 1997-09-09 | 1998-05-11 | Formulation for reducing urea effect for immunochromatography assays using urine samples |
JP10249579A JPH11133023A (en) | 1997-09-09 | 1998-09-03 | Compound for reducing influence of urea on chromatographic immunoassay using urine sample |
DE69819833T DE69819833T2 (en) | 1997-09-09 | 1998-09-07 | Methods and test strips to reduce the influence of urea in immunochromatographic measurements using urine samples |
EP98116837A EP0902287B1 (en) | 1997-09-09 | 1998-09-07 | Method and test strip for reducing urea effect of immunochromatographic assays using urine samples |
AU83183/98A AU726976B2 (en) | 1997-09-09 | 1998-09-08 | Formulation for reducing urea effect for immuno-chromatography assays using urine samples |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/925,998 US6824985B1 (en) | 1997-09-09 | 1997-09-09 | Formulation for reducing urea effect for immunochromatography assays using urine samples |
Publications (1)
Publication Number | Publication Date |
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US6824985B1 true US6824985B1 (en) | 2004-11-30 |
Family
ID=25452576
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/925,998 Expired - Fee Related US6824985B1 (en) | 1997-09-09 | 1997-09-09 | Formulation for reducing urea effect for immunochromatography assays using urine samples |
Country Status (6)
Country | Link |
---|---|
US (1) | US6824985B1 (en) |
EP (1) | EP0902287B1 (en) |
JP (1) | JPH11133023A (en) |
AU (1) | AU726976B2 (en) |
CA (1) | CA2237269A1 (en) |
DE (1) | DE69819833T2 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060029926A1 (en) * | 1998-05-06 | 2006-02-09 | Metrika, Inc. | Blocking compositions for immunoassays |
US20070172963A1 (en) * | 2003-06-03 | 2007-07-26 | Krauth Gary H | Native analyte as a reference in lateral flow assays |
US20090162879A1 (en) * | 2006-08-21 | 2009-06-25 | Panasonic Corporation | Measuring device, measuring apparatus, and measuring method |
US9488663B2 (en) | 2013-03-14 | 2016-11-08 | Abbott Point Of Care Inc. | Electrochemical methods and devices for amending urine samples for immunosensor detection |
US9651547B2 (en) | 2013-03-14 | 2017-05-16 | Abbott Point Of Care Inc. | Electrochemical methods and devices for amending urine samples for immunosensor detection |
WO2022016071A1 (en) * | 2020-07-17 | 2022-01-20 | Trustees Of Boston University | Viral detection systems and uses thereof |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7935539B2 (en) | 2003-02-14 | 2011-05-03 | Siemens Healthcare Diagnostics, Inc. | Generic method for latex agglutination assays |
JP2006526788A (en) * | 2003-06-03 | 2006-11-24 | バイエル・ヘルスケア・エルエルシー | Verification apparatus and method for optical inspection equipment |
WO2007082545A1 (en) | 2006-01-19 | 2007-07-26 | Lattec I/S | Dry stick device and method for determining an analyte in a sample |
AU2007207241B2 (en) | 2006-01-19 | 2012-07-26 | Lattec I/S | A novel dry stick device construction and method for determining an analyte in a sample using said dry stick device |
CN102778559A (en) * | 2012-08-14 | 2012-11-14 | 广州万孚生物技术股份有限公司 | Colloidal gold immunochromatographic detection kit and preparation method thereof |
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1997
- 1997-09-09 US US08/925,998 patent/US6824985B1/en not_active Expired - Fee Related
-
1998
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- 1998-09-03 JP JP10249579A patent/JPH11133023A/en not_active Withdrawn
- 1998-09-07 EP EP98116837A patent/EP0902287B1/en not_active Expired - Lifetime
- 1998-09-07 DE DE69819833T patent/DE69819833T2/en not_active Expired - Fee Related
- 1998-09-08 AU AU83183/98A patent/AU726976B2/en not_active Ceased
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Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060029926A1 (en) * | 1998-05-06 | 2006-02-09 | Metrika, Inc. | Blocking compositions for immunoassays |
US20070172963A1 (en) * | 2003-06-03 | 2007-07-26 | Krauth Gary H | Native analyte as a reference in lateral flow assays |
US8101429B2 (en) | 2003-06-03 | 2012-01-24 | Siemens Healthcare Diagnostics Inc. | Native analyte as a reference in lateral flow assays |
US20090162879A1 (en) * | 2006-08-21 | 2009-06-25 | Panasonic Corporation | Measuring device, measuring apparatus, and measuring method |
US7960132B2 (en) | 2006-08-21 | 2011-06-14 | Panasonic Corporation | Measuring device, measuring apparatus, and measuring method |
US9488663B2 (en) | 2013-03-14 | 2016-11-08 | Abbott Point Of Care Inc. | Electrochemical methods and devices for amending urine samples for immunosensor detection |
US9651547B2 (en) | 2013-03-14 | 2017-05-16 | Abbott Point Of Care Inc. | Electrochemical methods and devices for amending urine samples for immunosensor detection |
US10209251B2 (en) | 2013-03-14 | 2019-02-19 | Abbott Point Of Care Inc. | Electrochemical methods and devices for amending urine samples for immunosensor detection |
US10234450B2 (en) | 2013-03-14 | 2019-03-19 | Abbott Point Of Care Inc. | Electrochemical methods and devices for amending urine samples for immunosensor detection |
WO2022016071A1 (en) * | 2020-07-17 | 2022-01-20 | Trustees Of Boston University | Viral detection systems and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
EP0902287B1 (en) | 2003-11-19 |
EP0902287A1 (en) | 1999-03-17 |
AU8318398A (en) | 1999-03-25 |
CA2237269A1 (en) | 1999-03-09 |
JPH11133023A (en) | 1999-05-21 |
DE69819833D1 (en) | 2003-12-24 |
AU726976B2 (en) | 2000-11-30 |
DE69819833T2 (en) | 2004-09-30 |
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